Living organisms function through the interactions of biomolecules intricately distributed in space and varying in time. Often, spatial variations within a tissue or cell hold the key to understanding the function of molecular components. The abundance of biomolecules can span a wide range. For example, protein concentrations range from millimolar (10−3 M) to attomolar (10−18 M), and perhaps less. Copy numbers as low as 101 to 102 protein molecules per cell have been reported. Although several analytical methods offer high sensitivity and spatial resolution (fluorescence measurement, voltammetric microelectrodes, etc.), the selectivity and specificity of these methods seldom allows for the unambiguous identification of biochemical species.
With the emergence of sophisticated mass spectrometric methods, such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry, identification and structural characterization of protein and other components has now become possible. These techniques offer excellent sensitivity (in certain cases down to attomolar) as well as detailed biochemical species information (e.g., protein identification including the detection of post-translational modifications).
In the mass-spectral analysis of biological materials, however, most spatial distribution information is lost during the sample preparation step, wherein cells are ground and thoroughly mixed to form a homogenized slurry which is then placed in a mass spectrometer for analysis. The conventional ESI source is not amenable to molecular imaging, as it requires a liquid sample. The situation can be improved by using MALDI, which involves applying a chemical matrix that is locally excited by laser light so that a plume of sample material is ejected from a focused spot on the sample. In principle, MALDI can be used to recover spatial distributions by collecting molecular information as the laser is rastered across the sample surface. However, three obstacles exist with the MALDI technique. First, the mixing and co-crystallization of the sample with the light-absorbing matrix largely obscures the original spatial distribution of analytes (e.g., through lateral mixing). Second, the need to transfer the sample into a vacuum environment for mass analysis considerably restricts the choice of samples. Significantly, both of these requirements for successful MALDI analysis exclude the possibility of in vivo measurements. The third obstacle associated with the MALDI technique is that the laws of physics and practical considerations limit the focusable size of the laser spot so that it is larger than the wavelength of the laser light, resulting in a laser spot larger than most cells of interest and, thereby, diminishing the value of MALDI in view of the need for sampling from smaller regions.
Thus, there remains a need in the art for an instrument which can not only identify peptides and proteins in a tissue or cell but which also provides information on their spatial and temporal distribution down to submicron resolution and which analyzes their activity in vivo.